Communication

Microfluidic Production of Alginate HydrogelParticles for Antibody Encapsulation andReleasea

Linas Mazutis,* Remigijus Vasiliauskas, David A. Weitz

Owing to their biocompatibility and reduced side ef

fects, natural polymers represent anattractive choice for producing drug delivery systems. Despite few successful examples,however, the production of biopolymer-based particles is often hindered by considerabletechnical challenges mainly associated with high viscosity of polymer fluids. In this work, we present a microfluidic approach for production ofalginate-based particles carrying encapsulated anti-bodies. We use a triple-flow microdevice to inducehydrogel formation inside droplets before theircollection off-chip. The fast mixing and gelationprocess produced alginate particles with a uniquebiconcave shape and dimensions approaching those ofmammalian cells. We show slow and fast dissolutionof particles in different buffers and record controlledantibody release over time.

L. Mazutis, R. VasiliauskasVilnius University Institute of Biotechnology, Vilnius LT-02241,LithuaniaE-mail: linas.mazutis@bti.vu.ltL. Mazutis, D. A. WeitzHarvard University, School of Engineering and Applied Sciences,Cambridge MA 02138, USA

aSupporting Information is available online from the Wiley OnlineLibrary or from the author.

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1. Introduction

Drug delivery systems based on natural biodegradable

polymers are attractive agents for improved delivery,

stabilizationandprolonged release of encapsulateddrugs.[1]

Inmany cases the techniques used for their production rely

on mechanical stirring[2] or spray-coagulation methods.[3]

These methods, although efficient, produce polydisperse

drug particles and give a poor control over their size and

shape. In this respect, droplet microfluidics provides a

valuable tool for encapsulation of various biologicals and

chemicals into highly monodisperse pico- and nanoliter

volume droplets.[4] Numerous examples reported to date

have exploiteddropletmicrofluidics to produce single[5] and

double emulsions,[6] as well as various types of particles

composed of responsive polymers[7] and biodegradable

materials.[8] In this context, alginate, a natural polysacchar-

idederivedfrombrownalgae, isanattractivebiomaterial for

different applications in medical sciences.[9] Due to their

excellent biocompatibility alginate-based hydrogels are

highly promising candidates for use as drug delivery

systems[10] or as biomedical implants.[11] Indeed, much

effort has been dedicated to producing different types of

alginate hydrogel droplets and particles. For example,

Huang et al., showed alginate droplet production using

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T-junction geometry, but the resultant bimodal distribution

required an additional step of separation.[12] Others, have

generated alginate droplets on-chip, while inducing an

‘‘external gelation’’ process in bulk.[13,14] Others, have

employed capillary devices to produce alginate-based

double emulsions,[15] Janus particles[16] and hydrogel beads

carrying encapsulated cells.[17,18] However, in all systems

reported to date alginate hydrogel particles are relatively

large in size (�50–200mm), thereby reducing the scope of

potential applications.[19,20] For example, the use of

microparticles as drug carriers requires their dimensions

to be reduced to those of a cell (�10mm). In particular, the

reduced size should facilitate prolonged circulation in

the blood and provide better encapsulation conditions of

single cells. Indeed, production of smaller particles

approaching the dimensions of a cell has been challenging

due to the relatively high viscosity of alginate solutions

(>10 cP) and poor control over the polymerization kinetics.

The latter difficulty is associated with fast gelation process

triggered by divalent ions,[21] therefore complicating

both, production of monodisperse droplets as well as

delivery of biological compounds into the droplets.[19,22] A

further requirement for drug-loaded particles is their

biocompatibility, showing no adverse interactions with

biomolecules, cells and tissues.

Here, we present a microfluidic approach for production

of alginate hydrogel particles of biconcave (pancake) shape

having the size resembling to those of mammalian cells

(�10mm). Using a microfluidic device with a characteristic

flow-focusing junction that facilitates the break-up of

viscous fluids into monodisperse droplets, we established

the conditions for robust production of homogeneous

alginate particles over extended periods of time. The size

and shape of particles was governed by the cross-section of

droplet stabilization channel: due to fast mixing and

gelation of the liquid streams the resulting microgel

particles acquired unique biconcave shape resembling

the red blood cells. Loading alginate particles with

fluorescently labeled antibodies allowed us to evaluate

their release over time in different aqueous buffers. Finally,

we tested the biocompatibility of alginate particles in the

whole blood sample derived from laboratory rodents.

2. Results and Discussion

2.1. Microfluidics Chip Design

Initially, we tested alginate droplet production following

principles reported previously,[19,22] however, we could not

recover alginate particles due to premature polymerization

on-chip. We also tested alginate particle production by

passively fusing alginate dropletswith droplets containing

Ca2þ ions,[16,23] but this approach led to poor control over

the monodispersity of the emulsion. Due to these

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difficulties, we aimed to create a simple and robust system

that would allow production of alginate particles over long

periods of time for encapsulation of biologically active

molecules such as antibodies or other therapeutic proteins.

Using soft lithography, we created a microfluidic chip

containing three aliquot inlets that merge into a single

channel upstream of the flow-focusing junction (FFJ) as

shown in Figure 1.

The channeldownstreamfromtheFFJwas1 000mmlong

to allow interface stabilization by surfactant before the

droplets collideandflowinto the collection tube.During the

course of droplet production, we noticed that viscosity

differences of three fluids trigger complex flow instabilities

resulting in polydisperse droplets. To prevent this from

happeningwe incorporatedfluid resistors for each aqueous

phase Rh¼ 19.2 mPa s � m�3), so that their hydrodynamic

resistances are considerably higher than that of the

collection channel (Rh¼ 3.2 mPa s � m�3).

In addition, we utilized a nozzle having a constriction of

10mmwide,which facilitated thepinch-off of viscousfluids

into monodisperese droplets. To validate the functionality

of the microfluidics device, we tested alginate droplet

production using different flow rates and alginate concen-

trations (Supplementary Figure S1). Stable droplet produc-

tionwasachievedup to2% (w/w)alginate, abovewhich the

viscosity of the alginate solution became too high making

the droplet production process unstable (Figure 1c).

To prevent premature hydrogel formation inside the

microfluidic chip, a stream of water was introduced

between the CaCl2 and alginate solutions. However, fast

diffusionof small Ca2þ ions throughthe thin layerofmiddle

stream could induce premature alginate polymerization

and eventually prevent droplet production. The distance of

transverse diffusion, x, between two laminar streams can

bedescribedas<x2>¼ 2Dt,whereD isdiffusionconstantof

a solute and t is a time that it takes for the solute from one

liquid streamtodiffuse intoa second stream.We found that

when using a microchannel of 80–100mm long and 10mm

deep, the middle phase had to be �5mm thick to prevent

premature alginate polymerization on-chip (Figure 1).

2.2. Alginate Droplet Production

Having established the design of a microfluidic chip, we

tested production of alginate droplets using different flow

regimes. Stable alginate droplet production was achieved

when the flow rate for the continuous phase was at least

two times higher than the total flow rate of the dispersed

phases. Using a total flow rate for the dispersed phase of

100–150mL �h�1, and for the continuous phase of

200–300mL �h�1, allowed reproducible alginate droplet

production over a period of a few hours. However,

independently of the flow regimes used, priming of the

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Figure 1. Design and operation of microfluidics chip. (a) The device consists of three inlets for, (1) polymer precursor (alginate), (2) waterphase, (3) initiator (CaCl2), (4) continuous phase, and (5) collection outlet. The channels for dispersed and continuous phases merge into asingle channel (red square), where droplet generation takes place. (b) Graphical schematics depicting droplet generation using threealiquots (alginate solution, water, and CalCl2) and oil (blue). (c) Droplet generation at different alginate and CalCl2 concentrations. Dropletgeneration is stable for hours of continuous operation when alginate and CaCl2 concentrations are below 2%; above this value dropletproduction becomes unstable due to fast polymerization and increased viscosity of alginate solution. Scale bars, 20mm.

Microfluidic Production of AlginateQ1 Hydrogel Particles. . .

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fluids was critically important. To prevent premature

alginate polymerization on-chip, and thus clogging of the

channels, themiddle stream (water) had to be first injected

into the chip and after the flow was stabilized (�3–5min),

infusion of alginate and CaCl2 solutions could follow.

We tested droplet production using different CaCl2concentrations ranging from 0.1 to 5% (w/w) dissolved in

water (Figure 1c). At the lowest concentrations tested (0.1%)

alginate droplets remained in a liquid form and did not

polymerize into hydrogel. By contrast, at CaCl2 concen-

trations between 2 and 5%, gelation occurred during the

droplet pinch-off process inducing the formation of a pearl-

like train (string), similar to the report previously.[24] In the

middle regime (0.5–1.0% CaCl2) polymerization occurred

within few milliseconds inside the droplets (immediately

after the pinch-off process) before their collection off-chip.

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Under these conditions, generation of monodisperse drop-

letswas stable over extendedperiods of time. Increasing the

total viscosity of the aqueous phase, however, induced

jetting and undesirable polydispersity of the droplets.

Using our system polydispersity (coefficient of variation

�20%) became pronounced when the alginate concen-

tration was higher than 2% (w/w) or when the total

viscosity of the aqueous phase reached �20 cP. The jetting

could be partly suppressed by using a mineral oil of higher

dynamic viscosity (�15 cP), yet reliable production of

particles was impossible due to pressure build-up and

collapse of the microfluidic device. The use of FC-40 carrier

oil (3.4 cP), however, was optimal allowing production of

monodisperse droplets over a period of a few hours.

Interestingly,whenusing1%alginatesolution theobtained

hydrogel particles had biconcave (pancake) shape

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Figure 2. The shape of alginate hydrogel particles. Alginateparticles produced with 10mm deep microfluidics chip usingthe flow rates: 30mL �h�1 for alginate 1% (w/w) solution,40mL �h�1 for H2O, 30mL �h�1 for CalCl2 and 300mL �h�1 forFC40 oil. Alginate particles were placed on a microscope coverslip and imaged under the bright field microscope. Thecharacteristic biconcave shape of resulting particles is clearlyvisible. Inset: scanning electron microscopy image of a singlealginate particle after 48h of incubation in physiological buffer.Note the disintegration of the alginate particle.

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resembling red blood cells (Figure 2). The characteristic

shape of resulting particles was mainly governed by the

cross-section of the channel connecting the nozzle with

outlet port (Figure1). Themicrodeviceused in thisworkhad

10mmdeep and20mmwide channel,which confines liquid

stream into pancake shape droplets. Due to fastmixing and

Figure 3. Antibody release from alginate-based hydrogel particles.suspended in 1mL water or 1mL of PBS buffer and the fluorescenceimage of alginate particles immediately after mixing with water.(c) Antibody release from alginate particles suspended in 1x PBS buffer.scale of antibody release. In phosphate buffered saline (PBS) buffer ca

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polymerization, the alginate stiffens rapidly into hydrogel

particles that get locked in a biconcave shape (Figure 2 and

Supplementary Figure S2). The size of such particles

approaches the dimensions of mammalian cells (�5mm

thick and �16mm wide) and therefore could find useful

applications in biomedicine and pharmaceutical sectors.

Although, the size of current particles precludes their wide

use for drug delivery applications relying on a micrometer

scale systems, yet the concept of ‘‘polymeric artificial cells’’

has been of interest not only as a means to deliver

encapsulated compounds but also as a tool tomodulate the

in vivo response.[25] Moreover, biocompatible particles,

having �15mm size, could also find useful applications for

in vivo delivery of the target cells (�6–8mm in size) or large

bio-macromolecules (e.g., antibodies). In contrast, theuse of

larger (�30mm) particles in vivo would be hardly possible

due to clogging of the blood vessels. The use of cell-size

particles as delivery systems could also benefitmany other

biomedical applications such as encapsulation of beneficial

microorganisms,[26] preparation of tumor vaccines,[27] or

triggering controlled immune response.[28]

2.3. Encapsulation and Release

Alginate hydrogel particles suspended in water shows a

delayed dissolution over the timewindow of 48h (Figure 2,

inset). This slow disintegration provides an attractive

option to prolong the release of encapsulated biologicals

and potentially enhance their therapeutic effect. We tested

the release of FITC-labeled mouse IgG antibodies in more

details. Droplets carrying encapsulated IgG were collected

off-chip and after emulsification was complete, alginate

particleswere released fromtheemulsionbymixing itwith

20% PFO (Figure 3a). The resulting particles were then

Alginate particles (n� 103) carrying FITC labeled antibody weredecay of individual particles monitored over time. (a) Fluorescence(b) Antibody release from alginate particles suspended in water.The error bars denote standard deviation. Note the difference in timelcium forms ionic pair with phosphate that loosens hydrogel matrix.

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Microfluidic Production of Alginate Hydrogel Particles. . .

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dissolved in phosphate buffered saline (1� PBS) or pure

water, and IgG release monitored over time with a

fluorescence microscope. Based on existing literature data,

our hydrogel particles have a pore size of �16nm[29] and,

therefore, the release of larger proteins (e.g.,�20nm in size)

should be slowed in time. As expected, alginate particle

dissolved in water slowly release encapsulated IgG over

36h (Figure 3b). In contrast, antibody release in 1� PBSwas

immediate (Figure 3c), presumably due to ion pair

formation between Ca2þ and PO42� groups resulting in a

loose hydrogel matrix and thus fast IgG release. In

mammalian serum, however, the free calcium concen-

tration is�100mg/mL�1:[30] an amount that would further

reduce the alginate hydrogel dissolution. Finally, we tested

the biocompatibility of alginate particles within biological

fluids by mixing alginate particles (n� 103) carrying

encapsulated antibodies with whole blood extracted from

a mouse.

Alginate particles remained well dispersed in the

whole blood with no signs of cell adherence to the

surface of particles or of causing undesirable blood

clogging (Figure 4). These results indicate that alginate

particles are biocompatible and might be well suited for

future biomedical applications. Subsequent research is

necessary, however, for evaluation of release kinetics of

antibodies from alginate particles into blood and slow

particle dissolution, and our current efforts are dedicated

to solve these tasks.

Figure 4. Alginate particle dissolution in mouse blood. Alginateparticles containing encapsulated mouse antibody IgG werediluted in mouse blood and imaged under bright fieldmicroscope. Mouse erythrocytes (dark biconcave ellipses,4mm in diameter) and alginate particles (light-grey biconcaveellipses, �15mm in diameter) remained well dispersed in ablood over the period of few hours without noticeableformation of agglomerates. Inset: single alginate particlecontaining encapsulated FITC-labeled antibody.

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3. Conclusion

We have described a microfluidic approach that allows

reliable production of alginate particles of unique,

biconcave shape and sizes. By controlling the volume of

alginate solution entering the droplet, we demonstrated

the production of monodisperse biconcave particles

approaching the dimensions of mammalian cells. Using

triple flow microdevice, we have encapsulated antibodies

and confirmed particle biocompatibility within blood

samples. Due to slow degradation of alginate particles in

aqueous fluids, the release of therapeutic compounds

should be extended, therefore, providing a means to

increase the biological action of therapeutics. We believe

that the microfluidics approach provided in this work will

be a useful addition to the existing tools for production of

biopolymer-based drug carriers. Further reduction of

alginate particle size down to 1–2mm size would be

another useful step for development of natural and

biodegradable drug delivery systems.

4. Experimental Section

4.1. Materials and Reagents

Sodium alginate from brown algae (4–12 cP, 1% in H2O), CaCl2 and

Phosphate buffered saline (1� PBS) were from Sigma–Aldrich and

Roche. The water used in the experiments was deionized with a

Millipore Purification system (Milli-Q). Carrier oil was FC-40 (3M)

with 3% (w/w) fluorosurfactant. FITC-labeledmouse IgGwas from

Jackson ImmunoResearch. 1H,1H,2H,2H-Perfluorooctan-1-ol (PFO)

was from Fluorochem Ltd.

4.2. Microfluidic Device Design and Fabrication

Rectangular microfluidic channels 10mm deep were fabricated

usingsoft lithography.Briefly, the siliconwafer (UniversityWafers,

Inc.) was coated with SU-8 2005 photoresist (Microchem Corp.)

using a spin coater (Laurell) and exposed to UV light source (OAI)

through the photolithography mask to create a master. The

poly(dimethylsiloxane) (PDMS) mixture, containing PDMS base

and the curing agent at 10:1 ratio, was poured onto the master,

degassed and cross-linked at 65 8C for �12h. The PDMS layer was

then peeled off and access holes were punched with a 0.75mm

diameterbiopsypunch to createopenings for inlet andoutlet ports.

Themicrochannelswere sealedbybonding thePDMSslab toaglass

slide after activation with an oxygen plasma (PlasmaPrep 2; GaLa

Instrumente GmbH). The channels were treated with surface

coating agent (Aquapel) to make them fluorophilic and

subsequently flushed with nitrogen.

4.3. Microfluidic Device Operation

Weused amicrofluidic systemwhere the flow regimewas laminar

with low Capillary and Reynold numbers, Ca�10�4 and Re�10�2,

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respectively. Themicrofluidic device indicated in Figure 1 (see also

Supplementary Information) consists of three inlets containing

passive filters used to trap dust, followed by fluid resistors used to

damp fluid fluctuations arising during operation of the device. The

liquids from each inlet flow into a central channel of 80mm long

and 30mmwidewhere theymeet and flow in laminar flow, before

being encapsulated into droplets. Emulsification of fluids occurs at

flow-focusing junction, which has a short and narrow constriction

of 10mm wide and 10mm long. The collection outlet is used to

collect the droplets. The fluids were injected into the microfluidics

chip via PTFE tubing (0.56�1.07 mm2) connected to 1mL syringes

(Braun)and25-gaugeneedles (Neolus). Theflowratesof liquidsand

oil were controlled by syringe pumps (PHD 2000, Harvard

Apparatus). The flow rate for aqueous phase was in the range of

20–50mL �h�1andfor thecontinuousphase itwas100–300mL �h�1.

Emulsions were collected off-chip into a 1.5mL tube. Droplet

productionwasanalyzedwithahigh-speedcamera (PhantomV7.2)

mounted on a Nikon Eclipse Ti-U microscope.

4.4. Alginate Particle Release from Microfluidic

Droplets

Once droplet is generated the fast mixing between alginate and

calcium ions induces immediate alginate gelation, which results in

solid alginateparticle occupying�80%ofdroplet volume. To release

alginate particles from the emulsion PFO was added on top of the

emulsion toafinalconcentrationof10%(v/v)and incubatedatroom

temperature for 5min. The supernatant containing alginate

particles was then transferred into a second tube for analysis.

4.5. Antibody Release Measurements

To study the antibody release, the solidified alginate particles

(5mL,n�105) carryingFITC-labeled IgGwere suspended in1 500mL

phosphate buffered saline buffer (1� PBS, pH [7.4]) or MQ-water.

The room temperature was controlled by air conditioner at

22�1 8C. At selected time points, 10mL sample (�102 particles)

was sandwiched between the microscope slide and cover slip and

green fluorescence of the alginate particles was recorded

using Leica confocal microscope (TCS SP5) and 488nm laser. The

emission light at 532�20nm was recorded with PMT embedded

inside the microscope system. For each data point �20–30

randomly chosen alginate particles were analyzed using

Microscope imaging software by Leica Microsystems to extract

the mean fluorescence intensity and standard deviation values.

Themeanfluorescence intensity of the particles at incubation time

0 was set as 100% (y-axis in Figure 3), followed by subsequent

measurements at selected time points.

Acknowledgements: We are grateful to Dovile Dekaminaviciutefor supplying us with a sample of mouse blood. This work wassupported by Lithuanian Agency for Science, Innovationand Technology (MITA, uVesicles 31v-40). R.V. holds postdoctoralfellowship from the European Union Structural Fundsproject ‘‘Postdoctoral Fellowship Implementation in Lithuania’’(VP1-3.1-SMM-01).

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Received: June 10, 2015; Revised: June 22, 2015; Published online:DOI: 10.1002/mabi.201500226

Keywords: alginate particles; dropletmicrofluidics; antibody; drugdelivery system

[1] J. W. Yoo, D. J. Irvine, D. E. Discher, S. Mitragotri,Nat. Rev. DrugDiscov. 2011, 10, 521.

[2] K. A. Smith, J. M. Ottino, M. O. de la Cruz, Phys. Rev. Lett. 2004,93, 204501.

[3] J. Tu, S. Bolla, J. Barr, J. Miedema, X. Li, B. Jasti, Int. J. Pharm.2005, 303, 171.

[4] W. J. Duncanson, T. Lin, A. R. Abate, S. Seiffert, R. K. Shah,D. A. Weitz, Lab Chip 2012, 12, 2135.

[5] G. F. Christopher, S. L. Anna, J. Phys.DAppl. Phys.2007, 40, R319.[6] A. R. Abate, J. Thiele, D. A. Weitz, Lab Chip 2011, 11, 253.[7] L. Y. Chu, J. W. Kim, R. K. Shah, D. A. Weitz, Adv. Funct. Mater.

2007, 17, 3499.[8] Q. Xu, M. Hashimoto, T. T. Dang, T. Hoare, D. S. Kohane,

G.M.Whitesides, R. Langer, D. G. Anderson, Small2009, 5, 1575.[9] C. Alvarez-Lorenzo, B. Blanco-Fernandez, A. M. Puga,

A. Concheiro, Adv. Drug Deliv. Rev. 2013, 65, 1148.[10] P. Eiselt, J. Yeh, R. K. Latvala, L. D. Shea, D. J. Mooney,

Biomaterials 2000, 21, 1921.[11] K. Y. Lee, M. C. Peters, K. W. Anderson, D. J. Mooney, Nature

2000, 408, 998.[12] K.-S. Huang, Y.-S. Lin, C.-H. Yang, C.-W. Tsai, M.-Y. Hsu,

Soft Matter 2011, 7, 6713.[13] L. Capretto, S. Mazzitelli, C. Balestra, A. Tosi, C. Nastruzzi,

Lab Chip 2008, 8, 617.[14] W. Y. Chen, J. H. Kim, D. Zhang, K. H. Lee, G. A. Cangelosi,

S. D. Soelberg, C. E. Furlong, J. H. Chung, A. Q. Shen, J. R. Soc.Interface 2013, 10, 20130566.

[15] L. Liu, F. Wu, X.-J. Ju, R. Xie, W. Wang, C. H. Niu, L.-Y. Chu,J. Colloid Interface Sci. 2013, 404, 85.

[16] L. B. Zhao, L. Pan, K. Zhang, S. S. Guo, W. Liu, Y. Wang, Y. Chen,X. Z. Zhao, H. L. W. Chan, Lab Chip 2009, 9, 2981.

[17] W.-H. Tan, S. Takeuchi, Adv. Mater. 2007, 19, 2696.[18] V. L. Workman, S. B. Dunnett, P. Kille, D. D. Palmer,Macromol.

Rapid Commun. 2008, 29, 165.[19] M. Lian, C. P. Collier, M. J. Doktycz, S. T. Retterer,

Biomicrofluidics 2012, 6, 044108.[20] J. H. Xu, S. W. Li, J. Tan, G. S. Luo, Chem. Eng. Technol. 2008,

31, 1223.[21] T. Braschler, A. Valero, L. Colella, K. Pataky, J. Brugger,

P. Renaud, Anal. Chem. 2011, 83, 2234.[22] D. Saeki, S. Sugiura, T. Kanamori, S. Sato, S. Ichikawa, Lab Chip

2010, 10, 2292.[23] L. Mazutis, J. C. Baret, A. D. Griffiths, Lab Chip 2009, 9, 2665.[24] H. Zhang, E. Tumarkin, R. Peerani, Z. Nie, R. M. a. Sullan,

G. C. Walker, E. Kumacheva, J. Am. Chem. Soc. 2006, 128,12205.

[25] T. M. S. Chang, Nat. Rev. Drug Discov. 2005, 4, 221.[26] B. J. Kim, T. Park, H. C. Moon, S. Y. Park, D. Hong, E. H. Ko,

J. Y. Kim, J. W. Hong, S. W. Han, Y. G. Kim, I. S. Choi, Angew.Chem. Int. Ed. Engl. 2014, 53, 14443.

[27] W. A. Li, D. J. Mooney, Curr. Opin. Immunol. 2013, 25, 238.[28] J. Kim, W. A. Li, Y. Choi, S. A. Lewin, C. S. Verbeke, G. Dranoff,

D. J. Mooney, Nat. Biotechnol. 2015, 33, 64.[29] K. I. Draget, G. S. Braek, O. Smidsrod, Carbohyd. Polym. 1994,

25, 31.[30] J. F. Sullivan, A. J. Blotcky, M. M. Jetton, H. K. J. Hahn,

R. E. Burch, J. Nutr. 1979, 109, 1432.

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